1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-02-25 to 2021-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

EC Number:

264-261-4

EC Name:

1,1'-[[3-(dimethylamino)propyl]imino]bispropan-2-ol

Cas Number:

63469-23-8

Molecular formula:

C11H26N2O2

IUPAC Name:

1-{[3-(dimethylamino)propyl](2-hydroxypropyl)amino}propan-2-ol

Test material form:

liquid

Details on test material:

Details described in study specific records

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): JEFFCAT® DPA catalyst
- lot/batch number: PFW190123
- Physical appearance: Liquid
- Manufactured date: 2019-03-04
- Retest date: 2022-06-03
- pH: 11,7
- Purity: This product is not specified by purity/assay but total titratable amine content. Amine content: 9.0 meq/g. Typical purity: 88.74 A%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC); Store under inert atmosphere, at temperature no higher than 22ºC.
- Stability and homogeneity of the test material in the vehicle under test conditions and during storage: The stability of the test item in the vehicle was established at 1 and 100 mg/mL under Eurofins Advinus Study No. G21131. Based on the results, the test item was stable in the vehicle (Milli-Q water) up to 24 hours when stored at room temperature and five days when stored at refrigerator (4°C to 8 °C).
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not specified
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): not specified

FORM AS APPLIED IN THE TEST: liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 96 female Wister rats, Hylasco Biotechnology (India) Pvt. Ltd. 4B, MN Park, Shameerpet Mandal, Turkapally Village, Medchal District, Telangana -500078
- Age at start of treatment: 13-14 weeks
- Weight at study initiation: Mean body weight; G1: 0.278 ± 0.017 kg, G2: 0.276 ± 0.016 kg, G3: 0.277 ± 0.019 kg, G4: 0.278 ± 0.019 kg
- Fasting period before study: not specified
- Housing: Rats were housed in standard polysulfone rat cages (size: Length 425 mm × Width 266 mm × Height 185 mm) with stainless steel top grill having facilities for pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Additionally, polycarbonate rat huts were placed inside the cage as environmental enrichment objects which were replaced once a week.
i. Pre-mating / Acclimatization: Two rats of the same sex per cage were housed.
ii. Mating: Female rats were cohabited with males in a 1:1 ratio in the same cage.
iii. Post-mating / Treatment: After mating confirmation, females were housed individually.
Nesting material (paper cuttings) were placed inside the cages from GD 18 onwards.
Steam sterilized corn cob was used as bedding material was changed along with the cage at least twice a week.
- Diet: ad libitum, Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany
- Water: ad libitum, Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was available in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After detailed clinical examination for good health and suitability for the study, 120 females and 100 male rats were acclimatized for five days before initiation of mating. Only nulliparous and non-pregnant females were used in the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23 °C
- Humidity (%): 57 - 66 %
- Air changes (per hr): 12 to 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-03-24 To: 2021-04-07

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q® Water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose formulations were prepared fresh daily before dosing and used within the established stability period.
- Required quantities of the test item was weighed in a beaker (previously calibrated to a desired volume*) for each dose levels separately and a small volume of vehicle (Milli-Q® water) was added and stirred using a glass rod till a uniform solution is obtained. The volume was made up to the mark using the vehicle to get the final desired concentration. The solution was mixed well by stirring using a magnetic stirrer.
- Pre-calibration of the beaker to desired volume: Milli-Q® water was measured in a graduated measuring cylinder to the final volume of the batch size (Example for initial dose formulation preparation, the batch size was 30 mL, Milli-Q® water was taken till 30 mL mark in the graduated measuring cylinder). The measured Milli-Q® water was transferred into the clean beaker (to be pre-calibrated) and upper and lower meniscus of Milli-Q® water was marked on the beaker using a glass marker. Once these lines were marked, the Milli-Q® water was discarded and the beaker was dried. These lines (upper) were used to make up to the volume while dose formulation suspension preparation.
- For vehicle control group, Milli-Q® water was used for dosing.
- Storage temperature of food: not specified

VEHICLE
- Concentration in vehicle: 0, 12.5, 35, 50 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for active ingredient analysis were collected from the dose formulations intended for first treatment and last treatment.

The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as backup set which was stored at ambient condition. For each set, composite samples were drawn and in six replicates from each preparation and in case of control duplicate samples was drawn. The collected samples were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the concentration of the dose formulation.
The analysis was carried out as per the method validated under Eurofins Advinus Study No.: G21131.

Formulations were considered acceptable as the mean results are within
±15.0 % of the nominal concentration and the relative standard deviation
(% RSD) as less than 10 %.

Details on mating procedure:

During the mating period, female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day. If sperm was detected in a vaginal smear and or if a vaginal plug was observed in the morning, the female was considered to be mated. This day was regarded as GD 0. The females were cohabited in batches of required numbers. This procedure was continued until there were sufficient numbers of GD 0 pregnant rats for the study. GD 0 pregnant rats obtained on each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software. After grouping and ascertaining the group mean body weight, the rats were given accession number as applicable to the group on each day of assignment.
Note: After confirming mating, females were housed individually, and the unselected females were euthanized. The males were disposed after mating procedure.

Duration of treatment / exposure:

14 days (GD 6 to GD 19)

Frequency of treatment:

at approximately the same time each day (varying by ± 3 hours).

Duration of test:

20 days (GD 0 to GD 20)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

24 /group (4 groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 125 (G2), 350 (G3) and 500 (G4) mg/kg/day were selected for this study based on the results of available OECD 422 study conducted by sponsor where NOAEL was considered to be > 500 mg/kg bw/day (nominal dose received) for male and systemic toxicity and maternal systemic toxicity, and greater then 500 mg/kg bw/day for embryo-fetal toxicity. During the study signs such as dyspnea and respiratory sounds were observed at 500 mg/kg bw/day. In addition to the test doses, vehicle control group was included.
- Rationale for animal assignment (if not random): The rat is selected because:
The rat is the most relevant and preferred rodent species for teratogenicity assessment as the test facility has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. The animal model has been proved to be susceptible to the effects of developmental toxicants.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for (rat) dam blood sampling: not speficied

Examinations

Maternal examinations:

MORTALITY
- Morbidity and mortality: twice daily during treatment period for i.e., once in the morning and once in the afternoon.
- Based on the lack of clinical signs, the observation for morbidity and mortality was carried out once during weekends and public holidays.

CAGE SIDE OBSERVATIONS: Yes
- Clinical signs: once daily during acclimatization period, twice a day (pre and post- dosing) during treatment days and at least once on other days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical signs: once daily during acclimatization period, twice a day (pre and post- dosing) during treatment days and at least once on other days.

BODY WEIGHT: Yes
- All females included in the study were weighed on gestation days 0, 3, 6, 9, 12, 15, 18 and 20.
- Intermittent body weight gains were calculated from GD 0–3, 3–6, 6–9, 9–12, 12–15, 15–18, and 18–20. Further body weight gains for period GD 0–6, GD 6–20 and GD 0–20 was derived and analyzed only for rats pregnant at caesarean section.
- The corrected body weight was obtained by subtracting the unopened uterine weight from terminal body weight (body weight on GD 20).
- The corrected body weight gain was calculated by subtracting the body weight on GD 6 from the corrected body weight.


FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- About 200 g of food (food input) was provided on GD 0. The food output was recorded and replenished to about 200 g on GD 3, 6, 9, 12, 15 and 18. Prior to terminal sacrifice, food left over was recorded on GD 20.
- The quantity of food consumed by each female was calculated for the following intervals: gestation days GD 0–3, 3–6, 6–9, 9–12, 12–15, 15–18 and 18–20. Further food consumptions for period GD 0-6, GD 6-20 and GD 0-20 were derived. Food consumptions were statistically analyzed and presented only for rats found pregnant at caesarean section. No spillage of food was observed during the food measurement intervals.

WATER CONSUMPTION AND COMPOUND INTAKE : No


POST-MORTEM EXAMINATIONS: Yes
- Prior to caesarean section, code numbers were generated for blindfolding the animal numbers in order to avoid bias during caesarean section and subsequent fetal evaluation. The animal code was written on the tail by striking out the permanent accession number.
- Sacrifice on GD 20; all rats were sacrificed (caesarean section) under isoflurane anaesthesia and gross pathological changes in placenta and in all visceral organs of rats were examined
- gross pathology: involved external observation and examination of thoracic and abdominal viscera including uterine contents which were performed on all rats in the study sacrificed at term (caesarean section).
- At necropsy, the thyroid gland was collected and weighed from each rat (post-fixation) and subjected to histopathological examination. The tissue was processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. Remaining unused tissue were archived.

OTHER:

Hormone Analysis:
- Parameters: Total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormone levels
- blood collection: before caesarean section, from each rat under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture. Blood samples (about 1 mL from each rat) were collected in plain labeled tubes and kept on bench top for 90 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labeled plastic tubes and stored at below -70°C until they were analysed.
- The thyroid hormones were estimated in serum by Enzyme Linked Immuno Sorbent Assay (ELISA) method using BIO-RAD microplate washer and BIO-RAD model 680 readers.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Pregnancy status, Gross evaluation of placenta

- Immediately after termination, the gravid uterus along with the cervix was excised, weighed, and immediately examined
- Uteri from rats that appeared non-gravid were subjected to 10% ammonium sulphide staining to observe implantation sites if any (identified as pregnant rats) or to confirm the non-pregnant status.

Blood sampling:

- Before caesarean section, the blood was collected from each rat under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture for the determination of total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormone levels.
- Blood samples (about 1 mL from each rat) were collected in plain labeled tubes and kept on bench top for 20 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labeled plastic tubes and stored at below -20°C until they were analysed.

Fetal examinations:

- On GD 20, the individual fetuses were delivered by hysterectomy and removed sequentially. The fetuses were euthanized using intraperitoneal injection of sodium thiopentone. Fetuses were subjected to external and visceral or skeletal examination.
- The following litter endpoints were investigated:
- Total number of fetuses,
- Number of live fetuses,
- Individual fetal body weight (g)
- Anogenital distance of all live rodent pups
- Sex of the fetus - external determination based on anogenital distance that was confirmed based on gonadal examination (internal sex) during visceral examination.
- Fetuses were examined for external and visceral or skeletal alterations and the observations were classified as variants, anomalies and malformations as defined below:
- Variants: An incidence distributed throughout a population in consistently large percentage including the ossification variations.
- Anomaly: Alteration in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal.
- Malformation: Structural anomalies that alter general body conformity, disrupt or interfere with normal body function, may have a detrimental impact on postnatal life, or may be incompatible with postnatal survival.

- External examinations: Yes: [all per litter ]

- Soft tissue examinations: Yes: [half per litter ]
Fetuses from each litter were prepared for fresh tissue visceral organ evaluation. The fetuses subjected for visceral evaluation were decapitated and heads were stored in 70% alcohol in an individual bottles labeled with study number, code number and fetal number among total number of fetuses. Further heads were examined by sectioning using modified Wilsons Razor blade sectioning technique. After head evaluation, as no abnormalities were observed, fetal heads were discarded.

- Skeletal examinations: Yes: [half per litter ]
The remaining half of the fetuses of each litter were prepared for skeletal evaluation by wet skinning followed by evisceration and staining. Fetuses were fixed in 70 % ethyl alcohol, eviscerated, and dehydrated in 95 % ethyl alcohol; macerated in 1.5 % KOH and stained with saturated, aqueous Alizarin red S in Mall’s solution. The excess stain was removed in Mall’s solution and fetuses were cleared by passing through grades of glycerol with thymol crystals. Individual bottles containing fetuses for skeletal evaluation were labeled with study number, code number and fetal number among total number of fetuses. After the skeletal evaluation, fetuses were pooled dam-wise in bottles containing 100 % glycerol with crystals of thymol to prevent fungal growth. The bottles were labeled with study number, code number and number of fetuses evaluated among total number of fetuses.

- Head examinations: Yes: [half per litter ]

Statistics:

- Data captured using the ProvantisTM laboratory information management system (LIMS), parameters such as maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss , number of implantations, sex ratio, number of corpora lutea, early and late resorptions, hormone analyses (T4, T3, TSH) and the weight of thyroid gland data were evaluated using the Levene test for homogeneity of variances and the Shapiro-Wilks test for normality of distributions. Data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA). Data found to be nonhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
- Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.
- The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.
- The incidence of fetus and litter (incidence and percent) observations for skeletal observations were tested using Cochran Armitage trend test and pair wise comparison were tested by Fisher’s exact test for group association.
- All hypothesis testing was carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05

Indices:

no data available

Historical control data:

no data available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

All animals survived to planned death and there were no clinical signs attributed to the test Item.

Mortality:

no mortality observed

Description (incidence):

All animals survived to planned death and there were no clinical signs attributed to the Test Item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights were not affected at all the doses tested; however, the mean body weight gain was significantly lower during GDs 6-9 at 350 and 500 mg/kg/day, compared to vehicle control group. Corrected mean body weight gains at 500 mg/kg/day were significantly lower (-27%) when compared to the control group.

However, the body weight gain during GDs 6-20 was comparable to vehicle control group at all the doses tested.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

As compared to vehicle control, significant reductions in mean food consumption were noticed following treatment at 350 and
500 mg/kg/day, during GD 9 – 12 and the decrease ranged from -6 to -15%. Consequently, the net food intake during GD 6- 20 (-7%) was lower than control. The marginal decrease in food consumption at 500 mg/kg/day was not associated with decrease in body weights and hence considered treatment related non-adverse finding.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum Thyroid Stimulating Hormone (TSH), Thyroxine (T4) and Triiodothyronine (T3) did not show any significant differences which could be attributed to test item at the end of treatment period. Minimal variation observed in thyroid hormones were considered as incidental and the changes lacked dose correlation.
Thyroid gland weight was unaffected by treatment. No treatment-related histopathological effects were observed in the thyroid gland.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid gland weight was unaffected by treatment. No treatment-related histopathological effects were observed in the thyroid gland.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes in any animal at any dose levels.
There were no organs with macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related histopathological effects were observed in the thyroid gland.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

The maternal parameters comprising of gravid uterine weight and mean number of implantations, early and late resorptions, pre- and post-implantation loss and dams with resorptions at all the tested doses were statistically comparable to the vehicle control group. Gross evaluation of placenta revealed no findings.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The maternal parameters of pre- and post-implantation loss at all the tested doses were statistically comparable to the vehicle control group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The maternal parameters comprising of gravid uterine weight and mean number of implantations, early and late resorptions, pre- and post-implantation loss and dams with resorptions at all the tested doses were statistically comparable to the vehicle control group. Gross evaluation of placenta revealed no findings.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The maternal parameters of early and late resorptions at all the tested doses were statistically comparable to the vehicle control group.

Dead fetuses:

no effects observed

Description (incidence and severity):

The litter parameters comprising of total number of fetuses, number of live fetuses, male and female fetal weights and ano-genital distance were statistically comparable to vehicle control group at all the doses tested.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of non-pregnant rats were 1, 1, 0 and 2 at 0, 125, 350 and 500 mg/kg/day, respectively.

Other effects:

not specified

Details on maternal toxic effects:

Details on Caesarean section
- The Day 0 mated females were dosed from GD 5 to 19 and sacrificed on GD 20.
- A total of 24 sperm positive females were included in the vehicle control and each of the three treatment groups. The number of rats sacrificed at term was 24 in all the groups. The number of non-pregnant rats were 1, 1, 0 and 2 at 0, 125, 350 and 500 mg/kg/day, respectively.
- The total number of fetuses were 341, 337, 360 and 360 at 0, 125, 350 and
500 mg/kg/day, respectively. While all the fetuses were subjected to external examination, approximately half of the total fetuses namely, 164, 163, 174 and 175 fetuses were subject to visceral evaluation and remaining half, 177, 174, 186 and 185 fetuses were subjected to skeletal examination at 0, 125, 350 and 500 mg/kg/day, respectively.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The male and female fetal weights were statistically comparable to vehicle control group at all the doses tested.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live fetuses was statistically comparable to vehicle control group at all the doses tested.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The litter parameters comprising of total number of fetuses, number of live fetuses, male and female fetal weights and ano-genital distance were statistically comparable to vehicle control group at all the doses tested.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The total number of fetuses, number of live fetuses, and male and female fetal weights were statistically comparable to vehicle control group at all the doses tested.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The ano-genital distance was statistically comparable to vehicle control group at all the doses tested.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examinations revealed no teratogenic effects attributed to the test item.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examinations revealed no teratogenic effects attributed to the test item in any litter at any dose level. There was significant increase in incomplete ossification of sternum no. 6 in 500 mg/kg/day, Delayed/incomplete ossification is generally considered transient changes and these finding denote generalized growth delays which normally ossify late in gestation. It also does not have general predictive value for teratogenicity [Carney and Kimmel (2007)]. Hence this variation was not considered treatment related. Increased incidence of Rudimentary 14th rib was observed at 125 mg/kg/day was not considered as treatment-related as the same was not observed at higher dose groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed during visceral examination of fetuses of rats treated up to 500 mg/kg/day. Fetal heads in all dose groups were normal when subjected to Wilsons-Razor blade sectioning.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

Dose formulation analysis

The results were considered acceptable, as the mean per cent recovery was in the range, +/- 15% at each dose level and % RSD from six replicates at each dose level was less than 10.0%.

Dose level (mg/kg/day)	Claimed concentration (mg/mL)	Average calculated concentration(a) (mg/mL)	Average %recovery (a,b)	%relative standard deviation(a)
0	0.0	0.0	NA	NA
125	12.5	12.7-12.9	102-103	4.55-7.17
350	35	35.6-37.0	102-106	4.65-7.36
500	50	51.0-51.1	102	1.57-4.88

(a) results are the range of values determined from two occasions

(b) average % recovery was calculated from the claimed concentrations

NA - not applicable 

Applicant's summary and conclusion

Conclusions:

Based on the findings, under the test conditions and doses employed in this study, it is concluded that No- Observed- Adverse- Effect- Level (NOAEL) for maternal toxicity and developmental toxicity is 500 mg/kg/day as the maternal and litter parameters were not affected up to 500 mg/kg/day. Teratogenicity is 500 mg/kg/day as fetal external, visceral and skeletal examinations revealed no signs of teratogenicity in any of the tested dose levels up to high dose of 500 mg/kg/day.